Abstract Glioblastoma (GBM) is the most lethal form of adult brain cancers with a median survival of <15 months despite aggressive standard chemoradiation. GBM are formed by GBM stem-like cells (GSCs) - a major contributor to tumor recurrence and a natural focus for therapeutic development. There are two main reasons responsible for treatment failure: 1) high intra- and inter-tumor cellular and molecular heterogeneity with multiple subclones possessing distinct genetic determinants; 2) GSCs exhibit multiple redundant signaling pathways requiring simultaneous targeting of overlapping pathways. We have invented and biologically validated a novel tandem computational platform, GeneRep-nSCORE that integrates large-scale gene expression profiles with genomic changes to identify common founding alterations or master regulators of GSCs that span a large number, if not all, GSC subclones within and across GBM tumors. We discovered such a core set of four common master regulators in GCSs that are outstanding targets for clinical development. Expression of these four factors was sufficient to reprogram normal astrocytes to GSCs, whereas their depletion profoundly abrogated GSCs, and thus tumor development in vivo, in all eight lines of patient-derived GSCs of varied genetic and molecular backgrounds examined to date. The goal of this application is to develop a customized set of Adeno-associated virus (AAV)-based genetic tools to target the whole spectrum of GSCs (Phase I) for the purpose of delivering targeting constructs to deplete the four common master regulators responsible for malignant transformation and proliferation in GSCs (Phase II). The specific objectives of this proposal are: (i) using directed evolution and available combinatorial AAV capsid library, and for the first time, introducing a dynamic mode of administration of a library reagent over the time course of tumor progression, to greatly increase the probability of identifying novel AAV variants specifically targeting slow-, and fast-cycling GSCs in patient-derived xenograft models (PDX) (Phase I); (ii) To design and validate a panel of AAV vectors that express shRNAs targeting core master regulators of GSCs to identify leads for preclinical testing; (iii) To optimize modes of viral delivery, pharmacokinetics and pharmacodynamics parameters, and safety and toxicity in normal and PDX treated with lead targeting AAV cassettes; and (iv) Based on these results, tools and basic DMPK data created, to conduct preclinical efficacy studies in PDX treated with lead targeting AAV cassettes either alone or in combination with standard chemoradiotherapy (Phase II) to prepare for an investigative new drug application for clinical testing in patients with GBM, and for commercial development of this novel technology.